The Achievement of Shuttle Vector Techniques in Mammalian Cell Mutation Research

Abstract

Shuttle vector plasmids have made major contributions to the advancement of mutation research for the last two decades. I have been involved in the development of shuttle vector plasmids and their experimental protocols, and herein provide a chronicle of shuttle vector studies. In the late 1960s, in vitro mammalian cell culture became a common technique in cell biology. Geneticists established a method to detect gene mutation as 8-azaguanine or 6-thioguanine resistance after cells had been exposed to radiation or chemicals. The resistance of the cells was found to be due to a mutation of the hypoxanthine-guanine phosphoribosyl transferase gene; however, which base was changed in the gene remained di‹cult to determine until DNA sequencing techniques were developed. After the Sanger method of DNA sequencing became available to geneticists, the next issue was how to identify numerous base changes in mutants easily and rapidly. A breakthrough on this issue was the use of the shuttle vector plasmid pZ189, which was developed for practical applications by Seidman and colleagues. Along with the development of new molecular biology techniques such as polymerase chain reaction and automatic DNA sequencing, pZ189 has been modiˆed to several forms as adaptations to the new techniques. These shuttle vector plasmids remain useful tools to reveal what types of mutations are induced by newly identiˆed mutagens at the DNA sequence level. This article describes some of the contents of my JEMS award lecture in 2011.

Genotoxicity of formaldehyde: molecular basis of DNA damage and mutation

“…Since no previous reports had described that formaldehyde induces tandem base substitutions that would arise from intrastrand crosslinks, we adopted the shuttle vector plasmid pMY189 (Matsuda et al, 1995;Yagi, 2013) that can be treated with a high concentration of formaldehyde. The plasmid pMY189 can replicate in both human and E. coli cells, and has an ampicillinresistant marker gene and a mutation marker gene supF that can be selected as colored colonies on bacterial agar plates.…”